Introduction

Evasion of apoptosis is one of the hallmarks of cancer (1) and can be achieved by overexpression of antiapoptotic proteins. Inhibitor of apoptosis proteins (IAP), such as cellular IAP (cIAP) 1 and 2 and X-linked IAP (XIAP), are key regulators of antiapoptotic and prosurvival signaling pathways; XIAP directly inhibits caspases, whereas cIAPs prevent the formation of proapoptotic signaling complexes. This leads to suppression of apoptosis through both the extrinsic and intrinsic apoptosis pathways (2–4). Their deregulation, through amplification, overexpression, or loss of endogenous antagonists, occurs in various cancers and is associated with tumor growth and poor prognosis, making them attractive targets for anticancer therapy (5).

The IAPs are characterized by their baculovirus IAP repeat (BIR) domains, which mediate protein:protein interactions; some members of the family, such as cIAP and XIAP, also possess RING (Really Interesting New Gene) zinc finger domains with E3 ubiquitin ligase activity (6, 7). The antiapoptotic activity of XIAP is mediated by its direct binding to and inactivation of caspases 3, 7, and 9 via its BIR domains (8). IAP antagonists such as the endogenous second mitochondria-derived activator of caspases (SMAC), which is released from mitochondria on induction of apoptosis, bind to the BIR domains of IAPs and can disrupt interactions such as those between XIAP and caspase-9 (9). On binding to other IAPs (cIAP1 and cIAP2), SMAC induces a conformational change, which activates their E3 ligase function, leading to rapid autoubiquitination and proteasomal degradation (10).

In response to TNFα, cIAPs ubiquitinate RIP1, promoting the formation of complexes (e.g., complex I), which activate survival signaling through the canonical NF-κB pathway. Simultaneously, formation of the death-inducing signaling complex (DISC), which drives apoptosis, is prevented. Antagonism and subsequent degradation of the cIAP1/2 leads to the stabilization of NIK (NF-κB-inducing kinase), which activates the noncanonical NF-κB pathway, resulting in the production of multiple cytokines including TNFα. Removal of the cIAP1/2 also allows the DISC to form, leading overall to a switch in TNFα signaling from prosurvival to proapoptotic (11–15). This loss of cIAP1/2 combined with release of the XIAP-mediated block on caspases, which is essential for full activation of apoptosis, leads to a sustained proapoptotic effect in the presence of TNFα via the extrinsic apoptosis pathway. Tumors with sufficient levels of TNFα in their environment may, therefore, be particularly susceptible to IAP antagonism (16). In addition, the antagonism of XIAP-mediated caspase inhibition promotes apoptosis induced by stimulation of the intrinsic apoptosis pathway by agents such as chemotherapeutics or DNA-damaging agents (17). This suggests that cIAP1/cIAP2/XIAP antagonists can be used to promote apoptosis through both the extrinsic and intrinsic pathways.

IAPs have been therapeutically targeted by antisense oligonucleotides and antagonist small molecules (4). AEG35156, an antisense oligonucleotide targeted to XIAP, showed some evidence of clinical activity (18) and sensitized cancer cells to chemotherapeutic agents and TRAIL receptor agonists in preclinical models (3). The first generation of SMAC-mimetic IAP antagonists to enter the clinic, all containing alanine moieties and inherently cIAP-selective, have shown some activity in preclinical models (19–22), but thus far limited single-agent efficacy in clinical trials (3, 4, 23–26).

We have previously reported the identification of lead compounds with activity against cIAP1 and XIAP by fragment-based screening and structure-based drug design (27, 28). Here, we describe the discovery and characterization of ASTX660, an antagonist of cIAP1/2 and XIAP, which is currently being tested in a phase I–II clinical trial (NCT02503423). We hypothesize that such IAP antagonism may lead to improved efficacy as a result of the more effective activation of apoptosis provided by blocking cIAP1/2 while releasing the XIAP block on caspases.

Materials and Methods

Materials

ASTX660 as the hydrochloride salt was synthesized using a chemical procedure similar to that used for AT-IAP (27). The key step involved the coupling reaction between methyl-5-((R)-3-methyl-morpholin-4-ylmethyl)-piperazine-1-carboxylic acid tert-butyl ester and 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}ethan-1-one (see Supplementary Materials and Methods); purity was determined as greater than 95% by high-performance liquid chromatography. All other reagents were purchased from Sigma unless otherwise stated. BV-6 [(2S)-2-{[(2S)-1-[(2S)-2-cyclohexyl-2-[(2S)-2-(methylamino)propanamido]acetyl]pyrrolidin-2-yl]formamido}-N-{6-[(2S)-2-{[(2S)-1-[(2S)-2-cyclohexyl-2-[(2S)-2-(methylamino)propanamido]acetyl]pyrrolidin-2-yl]formamido}-3,3-diphenylpropanamido]hexyl}-3,3-diphenylpropanamide] (14) was purchased from Selleckchem.

Protein production and crystallography

A XIAP-BIR3 construct (amino acids 250-354) was expressed in E. coli, purified by affinity and size exclusion column chromatography, and crystallized at approximately 10 mg/mL as described previously (27). Crystals were soaked with 5 mmol/L ASTX660 in 5% DMSO overnight at room temperature prior to data collection. The crystals had cell dimensions of approximately 70 Å × 70 Å × 105 Å and belong to space group P4122. The diffraction observed ranged from 1.7 to 3.0 Å.

Binding assays

Interaction between ASTX660 and the BIR3 domains of XIAP or cIAP1 was determined by measuring the displacement of a fluorescent peptide tracer derived from SMAC (AbuRPFK(5&6FAM)-amide; Peptide Synthetics Ltd) by fluorescence polarization on a Pherastar plate reader (BMG Labtech). IC50 curves were generated using GraphPad Prism version 6 and fitted using the four parameter logistic curve fit.

Cell lines

The human cell lines MDA-MB-231 and HEK293 were purchased from the European Collection of Cell Cultures (ECACC); human melanoma cell lines, A375 and SK-MEL-28, were purchased from ATCC; and the diffuse large B-cell lymphoma cell line, WSU-DLCL2, was purchased from DSMZ. All were grown in DMEM medium supplemented with 10% FBS and maintained at 37°C in an atmosphere of 5% CO2 except WSU-DLCL2 cells, which were grown as above except in RPMI medium supplemented with 10% FBS. All cell culture reagents were purchased from Invitrogen unless stated otherwise. These cells lines were not passaged for more than 6 months (or 30 passages) after authentication by the cell bank (short tandem repeat PCR) and were routinely screened for mycoplasma (MycoAlert, LONZA). Melanoma cell lines screened at ChemPartner were purchased from ATCC, except COLO679 (ECACC), GAK (Japanese Collection of Research Bioresources), MMAC-SF (Riken Cell Bank), and normal human dermal fibroblasts (NHDF; LONZA), and were mycoplasma screened, short tandem repeat PCR verified, and not used beyond 10 passages.

Immunoprecipitation with anti-XIAP

Equivalent amounts of cell lysate were incubated overnight at 4°C with protein A/G magnetic beads (Pierce) coated with anti-XIAP polyclonal antibody (R&D Systems). The beads were washed and boiled in SDS sample buffer containing DTT, before analysis of the eluted proteins by Western blotting. Western blots of the same lysate before immunoprecipitation were used for comparison. Antagonism of XIAP by ASTX660 was monitored by Western blotting for levels of SMAC immunoprecipitated by XIAP.

An MSD plate-based assay was used to quantify levels of cIAP1 in MDA-MB-231 after 2-hour ASTX660 treatment. Cells were incubated with compound for 2 hours, washed, and lysed. Lysates were applied to MSD plates as described previously (27).

Live cell imaging

Cells were imaged in real time using the IncuCyte ZOOM live cell imager (Essen BioScience). Cells were incubated with compound in 0.1% (v/v) DMSO, with or without neutralizing anti-TNFα antibody (R&D Systems) for 5 days, and live images were taken every 3 hours using a 10× objective. IncuCyte software was used to calculate mean percent confluency from four nonoverlapping phase-contrast images of each well.

Induction of apoptosis was measured over the first 24 hours by including the Essen BioScience IncuCyte Caspase-3/7 Reagent at a final concentration of 2 μmol/L in all the wells. Apoptotic cells were identified by the appearance of green-labeled nuclei, and green fluorescence was measured in real time in the green FL1 channel of the IncuCyte ZOOM live cell microscope.

Apoptosis cytometry assays

After incubation of the cells with ASTX660 for the designated length of time, cells were harvested by trypsinization, spun down, and 100 μL FACS buffer (PBS + 1% FBS) was added. Cells were then added to a 96-well plate and 100 μL of 2× CellEvent reagent (Thermo Fisher Scientific; 4 μmol/L in FACS buffer) was added. The plate was incubated in the dark for 30 minutes before measuring fluorescent stained cells in a Guava easyCyte HT cytometer (Millipore). Cleaved caspase-3 staining was recorded in the FL1 channel, with unstained and DMSO control wells being used to set the gated stained and unstained cell populations.

Cell line viability screening

In-house cell viability assays were set up using alamarBlue reagent (Bio-Rad) as described previously (27). A human melanoma cell line panel was analyzed at ChemPartner by CellTiter-Glo luminescent cell proliferation assay (Promega) after ASTX660 treatment for 72 hours in the presence or absence of 1 ng/mL TNFα (R&D Systems). Data were normalized to 0.1% DMSO (v/v) control, and the drug response, measured as the area over the dose–response curve (activity area), was determined for each cell line (29).

In vivo studies

All mice were purchased from Envigo. The care and treatment of animals were in accordance with the United Kingdom Coordinating Committee for Cancer Research guidelines and with the United Kingdom Animals (Scientific Procedures) Act 1986 (30, 31). The study protocols were approved by the University of Cambridge Ethical Review Committee.

Initial pharmacokinetic studies were performed in male BALB/c wild type as described previously (27). ASTX660 was either dissolved in saline and administered intravenously at 5 mg/kg in a dose volume of 5 mL/kg or dissolved in water adjusted to pH 5.5 with NaOH and administered by oral gavage at 5 to 30 mg/kg in 10 mL/kg. Blood samples were collected at various time points and plasma prepared by centrifugation. Further pharmacokinetic and pharmacodynamic studies were performed using tumor-bearing immunocompromised animals (see below). Tumors were excised at specific time points after oral dose and immediately snap frozen in liquid nitrogen before being stored at −80°C prior to analysis.

MDA-MB-231 xenografts were prepared by subcutaneously injecting 5 × 106 cells, suspended in 100 μL of serum-free medium, into the right hind flank of male SCID (BALB/cJHan®Hsd-Prkdcscid) mice. A375 xenografts were prepared by subcutaneously injecting 5 × 106 cells, suspended in 100 μL of a 1:1 mixture of serum-free medium and Matrigel (approximately 10 mg/mL, Corning), into the right hind flank of male nude mice (BALB/cOlaHsd-Foxn1nu). Subcutaneous xenograft tumors of HEK293 expressing FLAG-tagged human XIAP and caspase-9 were prepared as described previously (27). Tumors were measured using digital calipers, and volumes were calculated by applying the formula for ellipsoid.

For tumor growth inhibition studies, tumor-bearing animals were randomized into groups of 7 to 8 with the average tumor volume of 100 mm3 (31 or 33 days after MDA-MB-231 cell injection and 19 days after A375 injection). Mice were randomized and oral ASTX660 treatment started on day 1. Control animals received water. During the treatment period, tumors were measured at least twice a week, and the effect on body weight was recorded daily where possible. Statistical analyses were performed using GraphPad Prism version 6. The effects of treatments were compared using one-way ANOVA and two-way ANOVA with Dunnett multiple comparisons test against vehicle control. Differences were deemed statistically significant when P < 0.05.

Analysis of tumor sample pharmacodynamic markers

Xenograft tumor lysates were prepared by grinding the frozen tissue to a fine powder with a mortar/pestle under liquid nitrogen, and then adding ice-cold lysis buffer [1% Triton X-100, 150 mmol/L NaCl, 20 mmol/L Tris·HCl pH 7.5, plus protease inhibitors (Roche), 50 mmol/L NaF and 1 mmol/L Na3V04], to the ground-up tumor powder. Samples were vortexed and left on ice for 30 minutes. Lysates were cleared by centrifugation, and samples of the supernatant removed for protein determination by BCA assay (Pierce).

For Western blotting, equivalent amounts of protein lysate had SDS sample buffer and a final concentration of 50 mmol/L DTT added, before being boiled, and analyzed by Western blotting as described above.

For immunoprecipitation assay of tumor lysates, equivalent amounts of xenograft lysate were incubated overnight at 4°C with protein A/G magnetic beads (Pierce) coated with anti-XIAP polyclonal antibody (R&D Systems) followed by Western blot analysis. For the MSD assay, equivalent amounts of xenograft lysate (200 μg/well) were incubated overnight at 4°C in a streptavidin MSD plate coated with biotinylated anti-XIAP polyclonal antibody (R&D Systems), before washing and probing with anti-SMAC, anti–caspase-9, or anti-XIAP antibody (Cell Signaling Technology) followed by the appropriate secondary detection antibody.

Pharmacokinetic analysis

Compound levels in plasma and tumor samples were measured and pharmacokinetic parameters were calculated as described previously with the exception of sample bioanalysis, which was undertaken using reverse-phase liquid chromatography-mass spectrometry (MS), using a Qtrap 4000 MS (AB Sciex), coupled to an Agilent 1200 HPLC system (Agilent) or a Quattro Premier MS coupled to an Acquity UPLC system (Waters; ref. 27).

Results

ASTX660 is a novel IAP antagonist that targets the BIR3 domain of cIAP1/2 and XIAP

A fragment screen (28), subsequent structure-based drug design campaign (27), and further optimization yielded ASTX660 (Fig. 1A), a potent, non-peptidomimetic, orally bioavailable, antagonist of cIAP1/2 and XIAP. ASTX660 potently inhibited the interactions between a SMAC-derived peptide and the BIR3 domains of XIAP (BIR3-XIAP) and cIAP1 (BIR3-cIAP1) with IC50 values less than 40 and 12 nmol/L, respectively. The X-ray crystal structure of ASTX660 bound to BIR3-XIAP protein (PDB 5OQW) revealed that this inhibitor binds to the surface of the protein by occupying the same 4 pockets (P1-P4) also recognized by the N-terminal sequence of the endogenous ligand SMAC (Fig. 1B) (32, 33). The piperazine ring occupies the P1 pocket, with the protonated nitrogen forming hydrogen bonds with the side chain of Glu314 and the backbone carbonyl of Asp309. The methyl substituent is in van der Waals contact with the side chain of Trp310 and hence efficiently fills a small lipophilic subpocket in P1. The P2 pocket is occupied by the morpholine ring, which stacks on the top of the central amide. The carbonyl of the central amide forms a hydrogen bond with the backbone NH of Thr308. ASTX660 extends into the P3 pocket with a 4-azaindoline bicycle, which forms further van der Waals contacts with the side chain of Trp323, the backbone carbonyl of Gly306, and the phenolic oxygen of the side chain of Tyr 324. Finally, the benzylic substituent grows from C-6 of the azaindoline into P4 (Fig. 1C).

ASTX660 is a novel antagonist of cIAP1/2 and XIAP. A, Chemical structure of non-peptidomimetic cIAP1/2 and XIAP antagonist, ASTX660, derived by fragment-based drug discovery. B and C, X-ray crystal structure of ASTX660 (in green) in complex with XIAP-BIR3 (PDB 5OQW). The Connolly surface of the protein in B is colored by electrostatic potential (red, negative; blue, positive; gray, neutral). Hydrogen bonds between ligand and protein in C are shown as dashed red lines.

ASTX660 potently antagonizes XIAP in cells

Given the potent binding of ASTX660 to the isolated BIR3 domain of XIAP, we investigated the ability of ASTX660 to antagonize the effects of XIAP in cells. Both caspases and SMAC bind to the BIR3 domain of XIAP in cells and so should be displaced by a XIAP antagonist. We measured the displacement of caspase-9 and SMAC in cells treated with ASTX660. To measure XIAP:caspase-9 binding, we generated a stably transfected HEK293 cell line in which full-length XIAP (FLAG tagged) and caspase-9 are overexpressed. This enabled us to observe the association of caspase-9 with XIAP. Two hours after addition of ASTX660 to this engineered cell line, the association between XIAP and caspase-9 was potently inhibited with an EC50 value of 2.8 nmol/L (Fig. 2A; Supplementary Table S1). To confirm the antagonism of endogenous XIAP by ASTX660, we also investigated the displacement of SMAC from XIAP in A375, melanoma cells. Cell lysates, treated overnight with a range of ASTX660 concentrations, were immunoprecipitated with anti-XIAP. A clear reduction in SMAC levels immunoprecipitated with XIAP was observed on treatment with concentrations of ASTX660 above 0.01 μmol/L (Fig. 2B). Furthermore, exposure times as short as 5 minutes to 1 μmol/L ASTX660 were sufficient to antagonize the interaction of SMAC with XIAP in A375 cells (Fig. 2C).

ASTX660 treatment leads to potent antagonism of XIAP in cells. A, An engineered HEK293 cell line overexpressing XIAP and caspase-9 was treated for 2 hours with indicated concentrations of ASTX660. XIAP:caspase-9 binding was measured by immunoprecipitation of FLAG-tagged XIAP and quantitation of XIAP-associated caspase-9 using an MSD plate–based assay. Results show the mean of duplicate values. B, A375 cells were treated for 16 hours with the indicated concentrations of ASTX660. Endogenous XIAP antagonism was measured by Western blotting cell lysates for SMAC levels bound to XIAP following immunoprecipitation with anti-XIAP (top). Total XIAP and SMAC levels were determined by Western blots of cell lysates before immunoprecipitation (bottom). C, A375 cell lysates were treated with 1 μmol/L ASTX660 for the indicated times. XIAP antagonism was measured by Western blotting for SMAC levels after immunoprecipitation with anti-XIAP (top). Total XIAP and SMAC levels were determined by Western blots of cell lysates before immunoprecipitation (bottom).

ASTX660 binds to and leads to the degradation of cIAP1/2 and stabilization of NIK in cells

Binding of an IAP antagonist to the BIR3 domain of cIAP1/2 induces a conformational change, which activates the protein's ubiquitin ligase activity from the C-terminal RING domain (34). This leads to rapid autoubiquitination and proteasomal degradation of cIAP1/2 (14). To demonstrate the antagonism of cIAPs by ASTX660, levels of cIAP1 were measured in the human breast cancer cell line, MDA-MB-231, after treatment with ASTX660. Measurement of cIAP1 using an immunosorbent assay MSD detection platform demonstrated that ASTX660 induced degradation of cIAP1, with an EC50 of 0.22 nmol/L after 2 hours of treatment (Fig. 3A; Supplementary Table S1). Further investigation demonstrated a similar effect in A375 cells where cIAP1 levels dropped rapidly after treatment with 1 μmol/L ASTX660 and remained significantly decreased for up to 48 hours after addition of the compound (Fig. 3B). The human diffuse large B-cell lymphoma cell line, WSU-DLCL2, which has high basal cIAP2, was used to demonstrate the effect of ASTX660 treatment on antagonism of both cIAP1 and cIAP2 over a range of times and concentrations (Fig. 3C). ASTX660 treatment of WSU-DLCL2 cells leads to significant cIAP1 and cIAP2 degradation after 1 and 4 hours. Levels of cIAP2 were restored by 24 hours possibly due to resistance previously observed with IAP antagonists after prolonged treatment in a high NF-κB signaling background (35). TNFα-induced levels of cIAP2 are also antagonized in A375 and SK-MEL-28 melanoma cell lines after ASTX660 treatment (see Fig. 4B).

Antagonism of cIAP1 by ASTX660 leads to its degradation in cancer cells. A, MDA-MB-231 cells were treated with the indicated concentrations of ASTX660 for 2 hours, lysed, and relative levels of cIAP1 measured using an MSD plate–based assay. Results show the mean of duplicate values. B, A375 cells were treated with 1 μmol/L ASTX660 for the indicated times and cIAP1 or XIAP levels analyzed by SDS-PAGE followed by immunoblotting. β-Actin was used as an internal control. C, WSU-DLCL2 cells were treated with the indicated concentrations of ASTX660 for the indicated times. Levels of cIAP1 and cIAP2 were analyzed by SDS-PAGE followed by immunoblotting. D, MDA-MB-231 cells were treated with the indicated concentrations of ASTX660 for 2, 6, 24, and 48 hours, lysed, and equal amounts of total protein were then analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies.

TNFα triggers ASTX660-induced apoptosis in cell lines. A, Viability (top) and apoptosis (bottom) were measured in breast cancer MDA-MB-231 cells after treatment with 0.1 μmol/L ASTX660 using IncuCyte ZOOM live cell imaging. Anti-TNFα antibody at concentrations of 1 or 10 μg/mL was added to neutralize TNFα in the cell supernatant to monitor effects on the cell survival. B, Melanoma cell lines, A375 and SK-MEL-28, were treated with 1 μmol/L ASTX660 plus or minus 1 ng/mL TNFα for 24 hours. Cells were then lysed, and equal amounts of total protein were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. C, A375 or SK-MEL-28 cells were treated with 1 μmol/L ASTX660 for 25 hours in the presence or absence of 1 ng/mL TNFα. Cleaved caspase-3 activity was measured by cytometry. D, Effect of 72-hour ASTX660 treatment on the viability of 33 melanoma cell lines [plus NHDF cells (*), included as a control]. Data generated by CellTiter-Glo in triplicate, in the presence or absence of 1 ng/mL TNFα, were normalized to 0.1% DMSO (v/v) control, and the drug response, measured as the area over the dose–response curve (activity area), was determined for each cell line (29).

Degradation of cIAPs leads to the stabilization of NIK and activation of the noncanonical NF-κB pathway. The effects of ASTX660 treatment on NIK levels and NF-κB signaling were also investigated. On treatment of MDA-MB-231 cells with ASTX660, the rapid degradation of cIAP1 was accompanied by a concomitant increase in the levels of NIK at 2 hours, which remained increased at 6 hours (Fig. 3D). The effect of NIK stabilization on noncanonical NF-κB signaling was further demonstrated by the depletion of NF-κB2 p100 and the increase in p52 after 24 and 48 hours of treatment with ASTX660. In addition, although canonical NF-κB signaling markers [phospho-p65 or NF-κB1 (p105/p50)] remained largely unaltered, levels of phospho-IκBα were rapidly increased posttreatment and remained above basal levels up to 48 hours after treatment (Fig. 3D). Together, these data suggest that ASTX660 binds to cIAP1 and 2, leading to their degradation and downstream effects on NF-κB signaling and apoptosis induction.

The mechanism by which ASTX660 induces apoptosis in cell lines is TNFα dependent

Effects of ASTX660 treatment on cell viability and apoptosis were investigated further in MDA-MB-231 cells. Overall viability of this cell line was reduced with an EC50 of 1.8 nmol/L (Supplementary Table S1), whereas cleavage of PARP and caspase-3, detected by Western blot 24 hours after treatment with ASTX660, indicated apoptosis was induced by ASTX660 treatment (Fig. 3D). These effects were investigated further by measuring cell viability and induction of cleaved capsase-3 in real time. Loss of viability was observed soon after treatment with 0.1 μmol/L ASTX660 (Fig. 4A), and this was reversed on addition of a neutralizing anti-TNFα antibody, indicating that the effects of ASTX660 were TNFα dependent and that this cell line produces autocrine TNFα levels sufficient for this ASTX660-induced response. The loss of viability was paralleled by an induction of apoptosis indicated by an increase in levels of cleaved caspase-3, an effect that, again, was reversed by the addition of the neutralizing anti-TNFα antibody (Fig. 4A; Supplementary Fig. S1).

Induction of apoptosis was further studied in two melanoma cell lines, A375 and SK-MEL-28. Although addition of 1 μmol/L ASTX660 to these cell lines led to a degradation of both cIAP1 and cIAP2, apoptosis was only observed in the presence of TNFα as demonstrated by an increase in the levels of cleaved PARP, caspase-3, and caspase-9, again indicating the dependence on TNFα (Fig. 4B; Supplementary Fig. S2). Activity of caspase-3, as a marker of apoptosis, was further investigated in these melanoma cell lines by measuring the cleavage of a caspase-3 substrate using flow cytometry. Treatment with ASTX660 or TNFα (1 ng/mL) alone showed no effect on caspase-3 activity, in either the A375 or SK-MEL-28 cells; however, when both treatments were combined, the activity of caspase-3 was greatly increased at time points up to 72 hours, indicating induction of apoptosis (Fig. 4C).

Effects of ASTX660 treatment on cellular viability were also investigated in a wider panel of 33 melanoma cell lines (Fig. 4D). Addition of TNFα increased or potentiated the inhibitory effect of ASTX660 for the majority of cell lines, but not necessarily for all, suggesting that at least in these cell lines, sufficient levels of endogenous TNFα are produced. NHDF cell viability was not significantly altered by ASTX660 treatment (Fig. 4D).

ASTX660 is orally bioavailable in mice and demonstrates prolonged antagonism of XIAP and cIAP1 in vivo

The pharmacokinetic parameters of ASTX660 were evaluated following intravenous and oral administration to mice. Following intravenous administration of ASTX660 at 5 mg/kg to male BALB/c mice, a moderate mean clearance of 41 mL/minute/kg was observed with a large volume of distribution of 2.7 L/kg. ASTX660 was orally bioavailable (34%) following administration at 10 mg/kg to mice.

ASTX660 distributed into MDA-MB-231 (Fig. 5A) tumor xenografts. ASTX660 was detectable in tumors up to 168 hours after a single oral dose of 20 mg/kg, and AUClast was 129 μmol/L/h/mL. In contrast, plasma exposure achieved an AUClast 14.3 μmol/L/h/mL and concentrations were below the limit of detection after 48 hours. A rapid reduction in cIAP1 levels coupled with antagonism of the XIAP:SMAC interaction was detected from 30 minutes and for up to 3 days following a single dose in the tumors in the same experiment (Fig. 5B). Further experiments with Western blotting analyses confirmed rapid cIAP1 degradation in MDA-MB-231 xenograft tumors after treatment and a concomitant induction of apoptosis within one hour as demonstrated by an increase in cleaved PARP and cleaved caspase-3 (Fig. 5C). A decrease in association of XIAP and SMAC was also observed in xenograft tumor tissue, again after one hour of treatment (Fig. 5D), indicating antagonism of XIAP by ASTX660. The antagonistic effects on XIAP were investigated further in a tumor model derived from HEK293 cells genetically engineered to express XIAP and caspase-9 (Supplementary Fig. S3). In this model, a single dose of ASTX660 disrupted the interaction between XIAP and caspase-9 or SMAC for at least 3 days. Tumor ASTX660 concentrations persisted over the duration of the study.

Orally administered ASTX660 treatment modulates pharmacodynamic markers in a MDA-MB-231 xenograft model. A, SCID mice bearing MDA-MB-231 xenograft were treated with a single 20 mg/kg dose of ASTX660 and compound concentrations in plasma or tumor samples analyzed. B, In the same tumor samples, levels of cIAP1 and XIAP:SMAC association were determined by MSD assay. C, Mice bearing MDA-MB-231 xenograft tumors received a single oral dose of ASTX660 at 30 mg/kg. Animals were sacrificed at the indicated time points, and protein levels in tumors were measured by immunoblotting of whole-cell lysates. D, In the same tumor lysates, XIAP:SMAC association was analyzed by anti-XIAP immunoprecipitation. Each sample represents individual animals. MSD assay values represent mean ± SEM from 3 animals.

The dose- and schedule-dependent antitumor effects of ASTX660 were investigated in the MDA-MB-231 breast cancer xenograft model (Fig. 6A and B; Supplementary Fig. S4A and S4B). Daily oral administration of ASTX660 at 5, 10, and 20 mg/kg significantly inhibited tumor growth (P < 0.05 from days 15, 18, and 11, respectively; Fig. 6A). An intermittent schedule (cycles of 7 consecutive days of dosing followed by 7 days of dose-holiday) at 20 mg/kg also achieved significant tumor growth inhibition, and its effects were equivalent to the continuous schedule in the same model (both P < 0.05 vs. vehicle treatment from day 8; Fig. 6B). The activity of ASTX660 was further investigated in an A375 melanoma xenograft model in nude mice (Fig. 6C; Supplementary Fig. S4C-i). Daily oral administration of ASTX660 at 10 and 20 mg/kg caused significant tumor growth inhibition in an A375 model (both P < 0.05 from day 8), although in vitro, this cell line was only sensitive to ASTX660 in the presence of TNFα in vitro (Fig. 4B and C).

Orally administered ASTX660 inhibits growth of MDA-MB-231 and A375 xenograft tumors. Each data point represents mean ± SEM from 8 animals unless otherwise indicated. A, ASTX660 was orally administered to SCID mice, bearing MDA-MB-231 xenografts, at 20, 10, and 5 mg/kg once daily for 25 days. Control animals received water. B, Comparison of the effects of varying ASTX660 administration schedule on MDA-MB-231 tumors. One group of tumor-bearing animals was treated with ASTX660 for 28 consecutive days (q.d., n = 7), and another was given 2 cycles of 7 consecutive days of dosing followed by 7 days of dose-holiday (q.d. × 7/no dose × 7). On dosing days, both treatment groups received ASTX660 once daily at 20 mg/kg. The unconnected symbols represent the dosing events (red square: q.d. and blue triangle: q.d. × 7/no dose × 7). The control group received water as vehicle once daily for 25 consecutive days. C, A375-bearing nude mice were orally treated with 10 or 20 mg/kg of ASTX660 for 14 consecutive days.

In all three studies, the ASTX660 treatments were well tolerated, and no excessive bodyweight loss or significant adverse effects were observed (Supplementary Fig. S4A-i, S4B-I, and S4C-i). It has been suggested that antagonizing multiple IAPs may lead to reduced tolerability in vivo (20, 36). To investigate the inflammatory effects of ASTX660, we tested the compound ex vivo on peripheral blood mononuclear cells (PBMC) from 2 healthy human donors. We observed no substantial evidence of increase in secretion of proinflammatory cytokines (TNFα, IL1β, IL2, IL6, IL8, IFNγ, and MCP-1) in a multiplexed cytokine assay (MSD) after a 48-hour treatment with 10 μmol/L of ASTX660 or with 10 μmol/L LCL161 (see Supplementary Table S2). This was in contrast to the bivalent antagonist, BV-6 (Genentech; ref. 14), which causes rapid cIAP1/2 depletion, potently antagonizes XIAP (37), and induced cytokine elevation in the same PBMC assays (Supplementary Table S2).

Discussion

Antagonism of IAPs is considered a promising therapeutic approach for activating apoptosis pathways in cancer, and a number of IAP antagonists have undergone evaluation in early clinical trials (38). However, despite several observations of objective clinical response, single-agent activity in the clinic has been limited (39). The N-terminal sequence (AVPI) of the endogenous protein SMAC provided the starting point for the first generation of peptidomimetic IAP antagonists. The AVPI peptide shows cIAP1 selectivity (∼200-fold over XIAP) due to differential interactions formed by the alanine residue in the P1 pocket of the BIR3 domains of XIAP and cIAP1. We have shown that this intrinsic selectivity is retained in the first generation of SMAC mimetics currently in the clinic (27). Given its function in inhibiting caspase activation, it has been proposed that XIAP antagonism plays a key role in the activation of apoptosis through both the intrinsic and extrinsic pathways (2, 40). An IAP antagonist with increased XIAP potency may lead to improved efficacy due to more effective activation of apoptosis provided by releasing the XIAP block on caspases (38, 45). An increased therapeutic window may also be achieved between efficacious dose and the onset of cytokine-induced toxicity that results from cIAP antagonism. Cytokine release syndrome has been reported as the dose-limiting toxicity in a phase I dose escalation study of cIAP-selective antagonist LCL161 (23), and such effects have also been described in detail in preclinical toxicity studies with another cIAP1/2 antagonist GDC-0152 (41). A true antagonist of cIAP1/2 and XIAP could better explore the clinical potential of IAP antagonism.

To develop such a compound, we have applied our fragment-based drug discovery approach (Pyramid™) to specifically identify nonalanine leads with a cIAP1–cIAP2–XIAP profile, avoiding the intrinsic selectivity resulting from peptidomimetic approaches based on the AVPI tetrapeptide (27, 28). A subsequent lead optimization campaign focused on reducing off-target activities and improving pharmacokinetic properties, resulting in ASTX660, a potent nonpeptidic IAP antagonist, structurally distinct from IAP antagonists previously reported. The resulting IAP antagonism of ASTX660 was demonstrated both in vitro and in vivo. ASTX660 binds to both cIAP1 and XIAP with low nanomolar potency, and this inhibition translates to cells, where potent cIAP1/2 antagonism was demonstrated by their degradation, which occurs on the induction of autoubiquitinylation upon antagonist binding; at the same time, antagonism of XIAP was measured by displacement of SMAC and casapse-9. Moreover, our data also confirm here that ASTX660 has potent long-lasting antagonism of cIAP1 and XIAP in vivo following oral administration accompanied by significant inhibition of xenograft tumor growth. In the assays used here, ASTX660 demonstrated potencies of 2.8 and 0.22 nmol/L, respectively, for XIAP and cIAP1 antagonism (12.7-fold difference). In contrast, other clinical stage monovalent SMAC mimetics do not achieve this level of potency for XIAP (range, 10–35 nmol/L) or this balance of antagonism (39-227 fold difference) under the same conditions (see Supplementary Table S1), making ASTX660 a novel compound for further investigating the effects of IAP antagonism.

There are several potential advantages to antagonizing XIAP in addition to cIAP1/2. Aberrantly high levels of XIAP have been linked to poor prognosis in a number of tumor types, including diffuse large B-cell lymphoma, renal cell carcinoma, bladder cancer, and colorectal cancer (42–45), while overexpression of XIAP in response to chemotherapy and radiotherapy has been proposed to contribute to resistance to these treatments (42, 46, 47). In addition, XIAP levels have also been implicated in modulating responses to immunotherapy, with increased levels in Hodgkin lymphoma preventing cytotoxic lymphocyte–mediated cytotoxicity by blocking granzyme B–induced apoptosis (48) and upregulation of XIAP in inflammatory breast cancer driving resistance to antibody-dependent cell-mediated cytotoxicity (49), further indicating potential for a molecule that potently antagonizes XIAP. Furthermore, the role of XIAP in both extrinsic and intrinsic apoptosis suggests that a potent XIAP inhibitor, such as ASTX660, might also be expected to show increased synergy with activators of both these apoptotic pathways, such as chemotherapeutics, radiotherapy, or TRAIL agonists, compared with a more cIAP-selective antagonist (2). It has been suggested that certain bivalent compounds that potently antagonize cIAP and XIAP are not well tolerated in vivo as they induce an excessive cytokine-mediated proinflammatory phenotype (20, 36). However, we have demonstrated that, unlike the bivalent compounds, ASTX660 does not elicit a toxic cytokine signature, suggesting that the IAP antagonist properties of this compound are not a disadvantage in this respect.

ASTX660 is currently being tested in a phase I–II study in subjects with advanced solid tumors and lymphomas (NCT02503423). Further studies with this novel compound will allow the true clinical potential of an IAP antagonist to be explored both as a single agent and in combination.

Acknowledgments

We warmly acknowledge scientific discussions with many Astex colleagues. We also thank Anne Cleasby for critical discussions and crystallographic support.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).